Aberrant activation of the sonic hedgehog (Shh) signaling network has been implicated in up to one- third of all human cancers and is a driver in one-fourth of all medulloblastoma cases. Suppressing Shh signaling thus provides a promising therapeutic target in oncology. Most known inhibitors of Shh signaling are selective antagonists of the pathway component, Smoothened (Smo). The first Smo antagonist was approved by the FDA last year. However, a major problem with these inhibitors is the emergence of drug resistance. We and others have shown that resistance to Smo inhibitors can arise due to activation of Shh signaling downstream of the drug target, Smo, as well as activation of other oncogenic pathways that can promote tumor survival. For example, we found that mutant HRAS(V12) activation simultaneously suppresses Shh signaling and instead shifts tumor cell dependence to MAPK/Akt signaling. Since Smo inhibitors are relatively new to oncology, the mechanisms of resistance have not been comprehensively covered and we predict that there are more examples of resistance to these drugs. We have established a novel in vitro cell culturing system to model Shh-dependent medulloblastoma that can be used to study response and resistance to treatments. In the proposed studies, we will employ genome-wide approaches to identify changes that confer resistance to Smo inhibitors and we will develop ways of selectively targeting tumors that have acquired resistance by focusing on critical survival mechanisms mediated by Shh activity. Aim 1: To identify novel mechanisms of resistance to Smo inhibitors. We will employ an in vitro forward genetic screen with transposon-mediated mutagenesis to identify drug-resistant mutations. To complement our in vitro studies and validate biological relevance, we will sequence the exomes of resistant tumors that arise from in vivo xeonografts of Shh-dependent medulloblastoma cells. Aim 2: To characterize tumor-specific survival signaling pathways in medulloblastoma. We will comprehensively define how the Bcl-2 family of apoptotic regulators is modulated by Shh signaling to regulate cell survival. We will also conduct a 500-compound small molecule screen in Shh- versus HRAS- dependent medulloblastomas to identify the critical components that are necessary for survival in these cells. We will then explore promising screen hits with in vitro and in vivo studies for targeted cancer therapies Smo inhibitors are currently in clinical trials for medulloblastoma and this work will be important for predicting and preparing for resistance cases as they arise. This work will also elucidate novel features of Shh survival signaling that can be targeted with next-generation therapies for medulloblastoma.